Our clinical studies have demonstrated that conditioning patients given combined kidney and hematopoietic cell transplants with total lymphoid irradiation (TLI) and anti-thymocyte globulin reliably induces mixed chimerism and tolerance to the transplanted kidney in HLA matched patients, enabling complete withdrawal of anti-rejection medications without cytopenias, graft versus host disease or other serious complications. Promising clinical studies indicate that this approach also induces persistent mixed chimerism in HLA haplotype matched patients, although withdrawal of anti-rejection medications has not yet been attempted in these chimeric patients. The goal of the proposed research is to determine the cellular and molecular basis for tolerance induction induced by TLI and anti-thymocyte serum (ATS) through studies in well-characterized mouse models. Our previous experiments in mice indicate that stable mixed chimerism and tolerance induced by TLI/ATS is dependent on the presence of both CD8+ antigen cross-priming myeloid DCs and type I invariant NKT cells, since tolerance cannot be induced in Batf3-/- and Ja18-/- recipients, which specifically lack cross priming DCs and invariant NKT cells, respectively. In addition, the conditioning regimen changes the balance of immune cells to favor CD8+ DCs and NKT cells, and results in their activation. Studies are designed to elucidate the effect of TLI/ATS conditioning on surface markers, cytokine secretion patterns and functions of the myeloid DC subsets and NKT cells, and how these effects impact the interactions between the DCs and NKT cells that promote tolerance. We will also analyze the role of donor bone marrow DCs in TLI/ATS tolerance induction, given the capacity of a subset of plasmacytoid DCs to suppress alloimmune responses. The proposed studies will lead to a detailed understanding of the mechanisms responsible for tolerance induction in TLI/ATS based conditioning, and thereby enable the development of enhanced clinical protocols for achieving tolerance in HLA mismatched organ and hematopoietic cell transplants.